Abstract

Background: Acute kidney injury (AKI) is common in hospitalized patients and is associated with increased morbidity, mortality, and cost. Currently, AKI is diagnosed after symptoms manifest; available diagnostic tests (e.g., serum creatinine, urine microscopy, urine output) have limited ability to identify subclinical AKI. Because of the lack of treatment strategies, AKI typically is managed with supportive measures. However, strategies exist that may prevent renal insults in critically ill patients; therefore, early recognition of AKI is crucial for minimizing damage propagation.

Summary: We discuss biomarker test characteristics, their strengths and weaknesses, and future directions of their clinical implementation.

Impact Statement

Acute kidney injury (AKI) affects millions of people in the US and increases morbidity, mortality, and healthcare expenditures. Available laboratory tests for early and accurate diagnosis of AKI have significant limitations. Novel biomarkers that predict development of AKI earlier are critically needed because they allow implementation of preventive strategies that potentially improve clinical outcomes. In this report, we review the most promising biomarkers of AKI that have been investigated during the past 2 decades and present a model for clinical implementation and areas for future investigation.

The Kidney Disease: Improving Global Outcomes (KDIGO)3 most recently defined acute kidney injury (AKI) as an increase in serum Cr by ≥0.3 mg/dL (≥26.5 μmol/L) within 48 h, to ≥1.5 times baseline within the prior 7 days, or a urine volume of <0.5 mL/kg/h for 6 hours (Table 1) (1). There are a broad range of etiologies of AKI in hospitalized patients, including decreased renal perfusion, intrinsic renal disease due to sepsis-associated tubular epithelial cell injury, glomerulonephritis, nephrotoxic effect of medications, and bladder outlet obstruction, among others. The incidence of AKI in hospitalized patients, with use of the KDIGO equivalent definition of AKI, ranges from 17% to 31%, depending on the sampled population (2–4). AKI is recognized as an important risk factor for progression to chronic kidney disease, accelerated progression to end-stage renal disease, cardiovascular disease, and congestive heart failure (3, 5). It is also associated with increased short-term morbidity and mortality and with annual healthcare expenditures estimated to be $6.6 billion to $10 billion in the US alone (6–10). The prognosis of patients with dialysis-requiring AKI has not changed in the past 50 years, and short- and long-term mortality rates still range from 40% to 80%, despite advances in extracorporeal renal replacement techniques (10). Although AKI is associated with highly detrimental outcomes, recognition of AKI is unacceptably delayed in up to 43% of hospitalized patients (11).

Considering the magnitude of the problem and known delays in diagnosis, early detection of AKI is crucial for instituting timely precautions that can potentially impact prognosis. The consensus definition of AKI has undergone substantial alterations to improve diagnostic criteria and allow early recognition. Further efforts have focused on using electronic health records for automated reporting of potential AKI to facilitate earlier clinical evaluation and possibly improve outcomes (12, 13). However, a major hindrance to early and accurate diagnosis is the low sensitivity and specificity of the conventional markers of AKI. Although currently AKI diagnosis is based on changes in serum creatinine and urine output, other tests including blood urea nitrogen, fractional excretion of sodium, and urine microscopy could be used to guide clinicians in determination of the extent and underlying cause of AKI. The use of creatinine as a marker of AKI has numerous limitations, including poor correlation with glomerular filtration rate during a dynamic state and variations in its production, secretion, and extrarenal excretion (14–16). Most importantly, creatinine is not a real-time biomarker, and its levels may not rise until after renal function is compromised, which could result in a missed therapeutic window. Urinary output, although a very sensitive and early marker of kidney dysfunction, is not a widely used criterion for AKI diagnosis due to difficulties in collection of urine output data, frequent use of diuretics, and issues related to indwelling urinary catheters (e.g., early removal of urinary catheter to avoid catheter-related urinary tract infection, catheter obstruction, lack of ability for automated collection of urinary output data with currently used devices). Other surrogate markers of kidney injury, including blood urea nitrogen, fractional excretion of sodium, and urine microscopy, have their own limitations with the latter 2 having very low sensitivity and specificity. Blood urea nitrogen levels vary inversely with glomerular filtration rate, however, and may be falsely increased in subjects with a high-protein diet, glucocorticoid therapy, tissue breakdown, gastrointestinal bleeding, total parenteral nutrition, and volume depletion, and falsely decreased in individuals with poor nutritional status or chronic liver disease due to decreased urea production.

To improve the outcomes of patients with AKI, real-time biomarkers must be identified that facilitate early diagnosis and expedite effective preventive and therapeutic measures. As such, the American Society of Nephrology has designated the identification and standardization of novel biomarkers of AKI a top priority. The Acute Dialysis Quality Initiative recommended early integration of biomarkers in the diagnosis of AKI, suggesting that earlier diagnosis can potentially improve outcomes (17). In the US, the only Food and Drug Administration (FDA)-approved biomarkers for early AKI prediction are the cell-cycle arrest biomarkers (insulin-like-growth-factor–binding protein 7 [IGFBP7] and tissue inhibitor metalloproteinases-2 [TIMP-2]; both described below); the platform to measure these biomarkers is commercially available both at the portable and central laboratory forms. This review aims to appraise the current literature on the detection and validation of biomarkers of AKI in various clinical settings.

Biomarkers of AKI

Universal attributes of an ideal biomarker are as follows: (a) is measurable by a rapid test of readily available samples (urine or blood); (b) is measurable by a cost-effective, biologically and physiologically plausible assay with high sensitivity and specificity; (c) has dynamic and rapidly changing levels that correlate with disease progression or improvement; and (d) has prognostic value (10, 15). Pioneering studies from the past 2 decades have identified candidate biomarkers of AKI with considerable potential in translational medicine. These biomarkers can be subdivided into 5 categories (Table 2) (15).

Neutrophil gelatinase-associated lipocalin

Neutrophil gelatinase-associated lipocalin (NGAL), also termed siderocalin or lipocalin-2, is a protease-resistant, 25-kDa polypeptide of the lipocalin superfamily, initially identified in human neutrophils (18). It is expressed in trace levels in various human epithelia, including kidney, trachea, lungs, stomach, and colon (19). It functions as a natural siderophore by scavenging the cellular and pericellular labile iron that is released from organelles during an ischemic or toxic insult. Reducing available catalytic iron inhibits bacterial growth and attenuates oxidative stress during organ injury (20). A decade after its discovery, an elegantly designed murine study that used a genome-wide interrogation strategy showed that NGAL was one of the maximally induced genes after initiation of renal ischemia. It was expressed in renal tubular cells and upregulated during an ischemic or toxic insult, and its use as an early urinary biomarker of AKI was suggested (21, 22).

In 2005, Mishra et al. (23) showed that NGAL was a robust early biomarker of subclinical AKI in pediatric patients after cardiopulmonary bypass; its elevation preceded any increase in serum creatinine by 1–3 days, and the increase was sustained throughout the duration of AKI. The area under the receiver operating characteristic curve (AUC-ROC) for prediction of AKI on the basis of the consensus AKI definition was 0.99 and 0.90 for urine and serum NGAL, respectively. A myriad of subsequent studies evaluated the diagnostic and prognostic value of urinary NGAL as a biomarker of AKI in various settings (20, 24–29). A multicenter prospective study of 1635 patients showed that a single measurement of urinary NGAL in the emergency department had better discriminatory ability to predict intrinsic AKI (AUC-ROC of 0.81) than other biomarkers (kidney injury molecule-1, urinary liver-type fatty-acid-binding protein, urinary interleukin-18, and cystatin C) (30). In a metaanalysis of 2538 patients (19 studies from 8 countries) that used a uniform creatinine-based definition of AKI (defined as Cr increase >50% within 7 days), the AUC-ROC of NGAL for predicting AKI across all settings was 0.83 (95% CI, 0.74–0.91) when using a median cutoff value of >150 ng/mL (31, 32). Table 3 summarizes subsequent metaanalyses showing the diagnostic value of NGAL in specific subgroups (33–37).

Ultimately, NGAL rises proportionally to the severity and duration of renal injury, is expressed very early after renal injury (within 1–3 h of renal insult and 36–72 h before any increase in creatinine), is obtained from a readily available source (urine or plasma), and can be rapidly assayed. The studies to compare the performance of NGAL in urine vs blood are scarce. While it appears both tests perform reasonably well, it seems urinary NGAL may carry higher specificity for the AKI. Its ability to predict AKI before clinical signs are evident can facilitate implementation of appropriate preventive measures and improve resource utilization. Although these characteristics make it a promising biomarker, its performance is discrepant in different settings, and existing studies have used varying cutoff values that result in varying diagnostic accuracy. In addition, there are conflicting data suggesting that age, race/ethnicity, preexisting renal disease, and sample source (urine vs plasma) may impact diagnostic accuracy. NGAL is released by activated neutrophils during bacterial infection and systemic inflammation and may increase in a graded manner with sepsis severity (38). Synthesis of the antimicrobial protein NGAL is dramatically increased in stimulated epithelia (including inflamed or malignant colonic epithelium), serum of patients with bacterial infection, sputum of patients with asthma or chronic obstructive pulmonary disease, and bronchial fluid from emphysematous lung (39, 40). As such, optimal cutoff values are unknown for these different clinical settings, especially given that interference with alternative “nonrenal” sources may diminish the test accuracy for diagnosing AKI. It is currently approved in Canada and Europe for clinical use (41, 42).

Kidney injury molecule 1

Kidney injury molecule 1 (KIM-1) is a type-1 transmembrane protein that contains immunoglobulin, highly O- and N-glycosylated mucin domains in its ectodomain, and a relatively short cytoplasmic tail (43). It is expressed in trace amounts in the kidney's proximal tubular cells under normal conditions, but its expression is dramatically upregulated in dedifferentiated proximal tubule cells after an ischemic insult (43, 44). The KIM-1 ectodomain is present in the urine after an ischemic insult and is stable for an extended period (45). Earlier studies showed increased urinary KIM-1 levels in patients with biopsy-proven ischemic acute tubular necrosis (as compared with other forms of AKI or chronic kidney disease) (44). This finding launched the development of rapid urine immunoassays and clinical studies exploring its utility in various settings, including cardiopulmonary bypass, cardiac catheterization, critical illness, and the emergency department (46, 47). In a cohort of patients admitted to the intensive care unit with normal renal function, KIM-1 detected stage 2 or 3 AKI within 12 h of sample collection, with an AUC-ROC of 0.69 (30). A recent metaanalysis evaluated the utility of urinary KIM-1 for the diagnosis of AKI across all settings—assay sensitivity was 74% (95% CI, 61%–84%) and specificity was 86% (95% CI, 74%–93%), with an AUC-ROC of 0.86 (95% CI, 0.83–0.89) (Table 3) (46). The authors of this metaanalysis found significant publication bias and considerable heterogeneity among different studies.

KIM-1 has clinical promise because of its ability to detect early subclinical tubular injury with a rapid and noninvasive test. However, diagnostic accuracy may vary depending on the type of assay used, patient age, clinical setting, cutoff used, timing of measurement, and other factors (46). It demonstrated better sensitivity when an enzyme-linked immunosorbent assay was used within 2–12 h of clinical insult, in cardiac surgery patients with acute ischemic tubular necrosis, and in infants or children as compared to adults (46). Its diagnostic and prognostic value needs to be further validated in larger trials, and optimal cutoff values for clinical use currently are undetermined. KIM-1 has been approved by the US FDA as a biomarker for preclinical drug development (48).

Interleukin 18

Interleukin 18 (IL 18) is a cytokine in the IL-1 superfamily. It is synthesized by some cells, including monocytes, macrophages, and proximal tubular epithelial cells, as a 23-kDa inactive precursor and is processed into an active 18.3-kDa cytokine by caspase-1 (49). After being induced in the proximal tubule, IL18 is released into the urine after cleavage by caspase-1 and can be measured in the urine by use of an enzyme-linked immunosorbent assay. In early animal models, Melnikov et al. (50) reported that kidney IL18 increases in the setting of ischemic AKI and induces tubular necrosis by mediating ischemia-reperfusion injury and neutrophil and monocyte infiltration of the renal parenchyma. In a subsequent study, urinary IL18 levels were shown to be markedly increased in human subjects with acute tubular necrosis compared with healthy subjects (51). The predictive accuracy of IL18 for AKI has since been studied in various clinical settings, including during cardiac surgery, in intensive care, after cardiac catheterization, and after organ transplantation. In a multicenter prospective study of 1635 patients, a single measurement of IL18 in the emergency department had poor discriminatory ability to predict intrinsic AKI (AUC-ROC, 0.64) compared with other biomarkers (uNGAL, uIL18, uLFABP, uCysC and uKIM-1) (30). In a 2013 metaanalysis that included various clinical settings, the sensitivity and specificity of IL18 to predict AKI was 0.58 and 0.75, respectively, with an AUC-ROC of 0.70 (Table 3) (52). IL18 may have better diagnostic accuracy in children and adolescents after cardiac surgery (52), but the metaanalysis was limited by high heterogeneity, different definitions of AKI, and different diagnostic cutoffs. Altogether, the studies suggest that urinary IL18 levels rise early in ischemic kidney injury (approximately 12 h before clinical AKI) and can be rapidly and affordably measured. The predictive accuracy of IL18 may differ depending on age (with better diagnostic accuracy in adolescents and children), time of obtainment, and reference range.

However, combining IL18 with other biomarkers improves its performance in predicting AKI. Further appropriately designed studies are warranted to elucidate the optimal time, cutoff, and setting in which IL18 would be a useful biomarker for AKI.

Liver-type fatty-acid-binding protein

Liver-type fatty-acid-binding protein (L-FABP), also termed FABP-1, is part of a family of 14- to 15-kDa cytosolic proteins that function in intracellular lipid trafficking and endogenous cytoprotection by reducing oxidative stress in ischemia-reperfusion. It is highly expressed in the liver but is also expressed in the kidneys, lung, pancreas, and intestine (53). In the kidney, L-FABP is expressed in the proximal tubules, where fatty acids are used as a primary source of energy metabolism (54). Free fatty acids are filtered through the glomeruli and reabsorbed into the proximal tubules, where they bind L-FABP and are transported to mitochondria or peroxisomes and metabolized through β-oxidation (55). The FABP14 gene is responsive to hypoxic stress, and a rise in urine L-FABP levels reflects the stress of proximal tubular cells (56). In a murine model of cisplatin-induced kidney injury, increased urinary L-FABP levels preceded a rise in creatinine by 72 h (57). In human studies, L-FABP was measured in the urine of patients immediately after reperfusion of living-related kidney transplant, and a significant correlation was observed between urine L-FABP levels and both peritubular capillary blood flow and ischemic time of the transplanted kidney (54). Among patients undergoing abdominal aortic aneurysm repair, preoperative urinary L-FABP levels in endovascular repair and 4 h after open repair predicted AKI with an AUC-ROC of 0.83 and 0.77, respectively (58). When combined with NGAL, L-FABP measured 0–2 h after cardiac surgery in adults predicted AKI with an AUC-ROC of 0.91–0.93 (59). A prospective pilot study of patients with chronic kidney disease undergoing coronary angiography showed that L-FABP levels in patients who ultimately had AKI were significantly higher, even before contrast administration, and were predictive of AKI with an AUC-ROC of 0.70 (60). Similarly, increased baseline L-FABP was linked with a high risk of AKI after an allogeneic stem cell transplant (61). In the critical care setting, urinary L-FABP predicted AKI with an AUC-ROC of 0.75 (62).

Urinary L-FABP levels, which can be tested with solid-phase enzyme-linked immunosorbent assay on the basis of the sandwich principle with a working time of 3.5 h, have a moderate predictive value for AKI development, with increased levels reflecting underlying ischemic tubular stress. Studies also demonstrate its ability to predict AKI even before an ischemic insult (60, 61). However, increased serum concentrations of L-FABP have recently been shown with obesity, insulin resistance, and high blood pressure, all in the absence of acute tissue injury (63, 64). Studies have also suggested it could be an early biomarker for lung damage in moderate acute respiratory failure and a diagnostic marker for nonalcoholic hepatic steatosis (65, 66). Although the contribution of serum L-FABP to urinary L-FABP may be minimal, as suggested by Kamijo et al. (67), the test needs validation across a spectrum of clinical settings to determine optimal cutoff values. It is currently approved in Japan for clinical use (41).

Tissue inhibitor of metalloproteinase 2 (TIMP2) and insulin-like-growth-factor–binding protein 7 (IGFBP7) are cell-cycle arrest proteins expressed in renal tubular cells during cellular stress (41). In addition to inhibiting matrix metalloproteinase, TIMP2 regulates the cell cycle by directly stimulating cell division or inducing G1 cell-cycle arrest, depending on the context (41, 68). IGFBP7 is a secreted protein and member of the IGFBP superfamily. It has also been implicated in the G1 cell-cycle arrest phase, which occurs promptly with cellular stress. The urinary test for the product of [TIMP2] [IGFBP7] was the first AKI biomarker approved by the US FDA after a series of studies revealed promising performance.

Four large premarketing studies were conducted; the first study (Sapphire study) involved a multicenter, international, heterogeneous sample of critically ill patients without evidence of AKI at enrollment. Published in 2013, the Sapphire study (69) was a 2-stage study with discovery and validation phases. The Discovery study recruited 522 critically ill adults with at least 1 risk factor for AKI development, and 340 candidate biomarkers were assessed for their ability to predict AKI. Urine TIMP2 and IGFBP7 were identified as the best-performing biomarkers, with an AUC-ROC of 0.75 and 0.77, respectively. The product of TIMP2 and IGFBP7 was superior to all previously described biomarkers, including NGAL, KIM-1, IL18, and L-FABP in that specific cohort, with an AUC-ROC of 0.80. The [TIMP2] [IGFBP7] assay was then validated in the Sapphire cohort. The primary end point was stage 2 or 3 AKI (Kidney Disease: Improving Global Outcomes criteria) within 12 h of enrollment; this end point was reached by 102 out of 728 patients (14%). The test demonstrated a high sensitivity and negative predictive value with a cutoff value of 0.3 (ng/mL)2/1000 and a high specificity with high positive predictive value with a cutoff value of 2.0 (ng/mL)2/1000. Another multicenter validation study, Opal, was performed; 154 critically ill adults were enrolled, and 27 had the primary end point (stage 2–3 AKI within 12 h) (70). At the 0.3 (ng/mL)2/1000 cutoff value, the [TIMP2] [IGFBP7] assay predicted AKI in the next 12 h with a sensitivity of 89% and a negative predictive value of 97%. At the 2.0 (ng/mL)2/1000 cutoff, the test's specificity was 95%, and the positive predictive value was 49%. Finally, the Topaz study further validated the use of [TIMP2] [IGFBP7] in critically ill adult patients; the primary end point was stage 2 or 3 AKI within 12 h of enrollment, as determined by 3 AKI experts (71).

In 2014, these studies led to US FDA approval of the test (NephroCheck; Astute Medical), which is a fluorescence lateral flow immunoassay of the urinary concentrations of [TIMP2] and [IGFBP7], displaying the result as the product of the 2 proteins as the AKI risk score [(ng/mL)2/1000]. The turnaround time of this test is only 20 min. It has since been studied in various settings, including cardiopulmonary bypass, high-risk surgery, and pediatrics (72–75). As with other biomarkers, the studies have reported varying accuracy, mainly due to heterogeneity in study design, cutoff values, and clinical settings, with AUC-ROC ranging from 0.70–0.97 (38). Limitations of the NephroCheck fluorescence immunoassay include the cost, interference by urine bilirubin (at concentration ≥72 mg/dL) and albumin (at concentrations ≥1250 mg/L), and the relatively rapid decline in levels after the initial renal insult (instead of a graded rise in levels with increasing severity of AKI) (41, 76). Currently, the point-of-care NephroCheck device costs approximately $5000, with the cartridge for 1 test being approximately $85. The cost–benefit of testing unknown given the field is yet in its infancy, and there have not been studies establishing that early recognition of AKI with use of these biomarkers will lead to reduced disease progression, morbidity, mortality, or total hospital cost (41). In hospitalized critically ill patients, a value of 0.3 (ng/mL)2/1000 is associated with 7 times the risk of developing moderate to severe AKI within 12 h of assessment (71). However, given the poor specificity at a cutoff value of 0.3 (ng/mL)2/1000, especially if used by nonexperts in a low-risk setting, false-positive results would be expected and could lead to inappropriate use of resources (i.e., nephrology consultation, renal ultrasound, additional laboratory evaluation).

Other markers

Other potential biomarkers of AKI include microRNAs, urinary low-molecular-weight proteins, and urinary tubular enzymes. All of these possible biomarkers require further study (Table 2).

Combining biomarkers to predict AKI

An approach that has gained more popularity is the use of a panel of biomarkers that differ in origin (within the renal parenchyma) to more accurately predict AKI. Biomarker panels have been developed for differentiation and early diagnosis of AKI, predicting the AKI progress, assessing its severity, and predicting mortality. In a prospective multicenter cohort study of 1219 adults and 311 children undergoing cardiac surgery, a panel of biomarkers (KIM-1, IL18, and NGAL, obtained at distinct time points postoperatively) improved discrimination of AKI compared with individual biomarkers (48). Similarly, the product of TIMP2 and IGFBP7 performed better than either entity alone when predicting AKI in critically ill patients (69). In adult patients undergoing cardiac surgery, a combination of NGAL and L-FABP (the latter measured 0–2 h after surgery) performed well, with an AUC-ROC of 0.91–0.93 (59). As our knowledge of the biologic roles of the identified biomarkers improves, specific panels may be developed that will help discriminate the potential underlying causes of AKI, progress of AKI, and overall prognosis (77). Table 4 provides a summary of each biomarker characteristics including their advantages and limitations when they are utilized in the clinical setting.

Clinical implementation of AKI biomarkers

Despite the progress that has been made in identifying AKI biomarkers, their use has not been widely accepted in clinical practice. Investigators from the 10th Consensus Conference of the Acute Dialysis Quality Initiative recommended incorporation of novel AKI biomarkers within the definition of AKI (17). They posited that use of biomarkers to determine the extent and location of injury could result in a more targeted and individualized treatment approach for each patient with AKI than with current AKI definitions, including the RIFLE (risk, injury, failure, loss, end-stage kidney disease) (78) or AKIN (Acute Kidney Injury Network) criteria (79) (Fig. 1).

When providers identify individuals with high AKI risk after clinical evaluation, biomarkers could then be used to further risk-stratify patients and efficiently direct resources toward preventing AKI development or its progression (Fig. 2).

Conclusion

The past century has seen a shift in the practice of critical care medicine from anticipatory to preventive. Early, biomarker-based identification of patients at risk of AKI development is a fundamental step toward AKI prevention. The present literature validating AKI biomarkers is heterogeneous in study design, in the optimization of the diagnostic standards, in the test cutoff values, in the time of obtainment, and in the clinical context; this heterogeneity makes immediate extrapolation of the data to clinical practice difficult. Moreover, the use of creatinine as the diagnostic reference criterion has major shortcomings because it does not detect subclinical AKI and may undermine the specificity of the tests. Additionally, the performance of certain biomarkers may be affected by specific clinical settings (e.g., the use of L-FABP to predict AKI in a patient with an underlying metabolic syndrome or nonalcoholic hepatic steatosis; or use of NGAL in patients with sepsis). The pathophysiology of AKI is very complex, with several distinct pathogenic mechanisms. As such, a single biomarker may not hold equal sensitivity and specificity compared with a distinct biomarker profile matched to the patient's predisposition and exposure history. As we gain further insights into the biologic function of biomarkers, development of biomarker panels to match the distinct disease processes will help improve test performance.

The future of AKI biomarkers remains exciting, but further steps are necessary before their clinical use is broadly accepted, including validation across a spectrum of settings and identification of discriminatory cutoff values. More research is needed to validate the benefits and cost-effectiveness of using biomarkers for early implementation of nephroprotective strategies in patients at risk. Early recognition of patients at risk of AKI will allow interventional studies to corroborate whether recognizing these patients and implementing early nephroprotective care will improve outcomes such as dialysis requirements and mortality rates.